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ICoN2 and the NCycle16 Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera Xi Li, Steven Parker, Manu Deeudom1 and James W. Moir2 Department of Biology, University of York, Heslington, York YO10 5DD, U.K. Abstract Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host–microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized. Introduction Parts of bacterial respiratory chains are, by necessity, embedded in the cytoplasmic membrane in order to allow generation of ion gradients to drive the synthesis of ATP. In Gram-negative bacteria, it is typical to also find redoxcofactor-containing respiratory proteins within the periplasm where they may function as electron carriers or redox enzymes. Many of the enzymes and electron carriers of, for example, denitrification are typically localized within the periplasm. A variant on this organizational structure is found in the genus Neisseria, in which a number of redox proteins are predicted to be associated with the outer membrane. The width of the periplasm (∼170 Å [1,2], where 1 Å = 0.1 nm) presents a potential problem for the movement of electrons from the inner membrane respiratory complexes to the outer membrane electron carriers/respiratory electronacceptor reductases. In the present paper, we review (i) the evidence for association of respiratory proteins in Neisseria with the outer membrane, (ii) the structural properties of the outer membrane respiratory proteins and the consequences of this for their functionality, (iii) additional possible roles for outer membrane attachment in the lifestyle of Neisseria species in their natural habitat, and (iv) the evidence that outer membrane-tethered redox proteins are also found outside the genus Neisseria. Key words: cupredoxin, cytochrome, electron transfer, Neisseria, periplasm, redox protein tethering. Abbreviations used: AniA, anaerobically induced protein A; CCP, cytochrome c peroxidase; GST, glutathione transferase; Laz, lipid-modified azurin; LCR, low-complexity region. 1 Present address: Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand. To whom correspondence should be addressed (email [email protected]). 2 Biochem. Soc. Trans. (2011) 39, 1895–1899; doi:10.1042/BST20110736 The outer membrane redox proteins of Neisseria The genus Neisseria (which belongs to the Betapreoteobacteria) consists of two pathogenic species (Neisseria meningitidis and Neisseria gonorrhoeae) and about 20 commensal species, which typically inhabit epithelial mucosal surfaces. Many are colonists of the human epithelial mucosa in the mouth and upper respiratory tract. Under oxygenlimited conditions, Neisseria species can denitrify nitrite [3,4], a process that relies on the expression of AniA (anaerobically induced protein A) [5–7], formerly known as Pan1 [8]. AniA is a copper-type nitrite reductase (NirK) [6,9], but is distinguished by being an outer membrane protein (based originally on selective solubilization of the cytoplasmic membrane using dodecyl sarcosinate [10]), rather than a periplasmic protein as is typical for NirK in other Gramnegative organisms. AniA was originally identified from N. gonorrhoeae [10], but is also present in outer membranes from other Neisseria species [11]. The association with the outer membrane is via a palmitate residue, which is covalently attached to the N-terminal cysteine residue that immediately follows the cleaved signal peptide [7]. Since localization of AniA was only determined biochemically using differential detergent-solubilization methodology, we set out to investigate localization of AniA in the commensal Neisseria lactamica, by isolation of intact membranes and separation by sucrose gradient centrifugation (Figure 1). The experiments provide useful confirmation that AniA [and Laz (lipid-modified azurin), see below] are found in the outer membrane, whereas the c-type cytochromes are associated with the inner membrane. C The C 2011 Biochemical Society Authors Journal compilation 1895 1896 Biochemical Society Transactions (2011) Volume 39, part 6 Figure 1 Localization of redox proteins in membranes in Neisseria N. lactamica cells were fractionated into periplasm, cytoplasm and membranes using lysozyme treatment and osmotic shock. Localization of proteins into the inner membrane (IM) or outer membrane (OM) fractions are shown in panels (A–D). (A) The major band from a Coomassie Blue-stained gel is a porin, only seen in outer membranes. (B) Western blot with anti-AniA antibodies shows that AniA is mainly associated with outer membrane. (C) Western blot with anti-Laz Figure 2 N. meningitidis Laz is enriched in outer membrane blebs N. meningitidis was fractionated into periplasm, cytoplasm and membranes using lysozyme treatment and osmotic shock, and outer membrane-containing blebs were prepared. Samples containing total cell extract (lane 1), blebs (lane 2), total membranes (lane 3), periplasm (lane 4) and cytoplasm (lane 5) are shown. Upper panel: Ponceau S staining; lower panel: probed with anti-Laz antibody. Molecular masses are indicated in kDa. antibodies shows that Laz is mainly associated with outer membrane. (D) Haem staining shows that c-type cytochromes are mainly associated with the inner membrane (48 kDa, CcoP; 30 kDa, cytochrome c5 /PetC; 24 kDa, CcoO/cytochrome c4 ; 16 kDa, cytochrome c’). In addition to a lipid-associated copper-type nitrite reductase, a Laz-like protein is found in Neisseria species [12– 14]. Azurins are copper-containing electron-carrier proteins found in many bacteria. The role of Neisseria azurin as an electron-transfer protein is unclear, but it may have an important role in the host–microbe interaction (see below). Like AniA, Laz consists of a signal sequence followed immediately by a cysteine residue which becomes covalently associated with outer membrane lipid. Using antibodies raised against Laz protein, we found that Laz is indeed associated with the outer membrane (Figure 1) or with outer membrane vesicles or blebs (Figure 2). Two other outer membrane-associated redox proteins are the CCP (cytochrome c peroxidase), which is found in N. gonorrhoeae, and not in most other Neisseria species [15], and the cytochrome c [16]. Again, homologous proteins from other genera are typically found as soluble proteins in the periplasm, but in Neisseria, they are associated with membrane. Flexible tethers for attachment to the outer membrane The Neisseria outer membrane respiratory proteins AniA, Laz and CCP share common organizational features. The N C The C 2011 Biochemical Society Authors Journal compilation terminal part consists of a predicted signal sequence, which is recognized by the globomycin-sensitive signal peptidase II [15]. This yields a mature peptide with an N-terminal cysteine followed by an extended LCR (low-complexity region), which is in turn followed by a protein domain with high identity with the globular domains of homologous nitrite reductase, azurin and CCP respectively. {We have excluded cytochrome c from this group, since (i) it appears to lack an LCR, and (ii) its location in the cell is questionable; although the Neisseria cytochrome c’ is an outer membrane protein when heterologously expressed in Escherichia coli [16], it appears to be associated with the inner membrane in N. lactamica and N. meningitidis (W.M. Huston, X. Li and J.W. Moir, unpublished work).} The LCR seems to be a Neisseria genus-specific addition to these proteins and consists of a stretch of 30–40 amino acids, rich in alanine (∼50% of the amino acid composition of LCR is alanine), proline, glutamate, glutamine, serine and threonine (Figure 3). The LCR in Laz is particularly similar to a repeated sequence motif (AAEAP) found in the outer membrane Lip protein, where the sequence is known as the H.8 antigen [17]. We predict that the LCR forms an extended unstructured region that acts as a flexible linker, attaching the globular domains of the proteins to the outer membrane, but enabling the proteins to be located at some distance from the membrane. It is not possible to be sure of the extension lengths of these linker/LCR domains in the absence of experimental support, but it would appear likely that the translational ICoN2 and the NCycle16 Figure 3 Putative flexible linker regions/LCRs from Neisseria outer membrane-associated redox proteins The regions shown begin after the predicted signal peptidase II cleavage site (which leaves an N-terminal cysteine residue) and end at the beginning of the predicted globular domain of the redox protein in question (deduced by comparison with homologous, but periplasmic, members of the same family). Amino acids alanine, glutamate, proline, serine and threonine are shown in bold to emphasize that most of the amino acids from the linker region belong to these types. donor to AniA [20,21]. We have argued previously that the tether regions of AniA should allow it to extend sufficiently from the inner leaflet of the outer membrane to form a complex with the inner membrane di-haem protein cytochrome c5 [21], so long as the tethers can extend up to ∼60 Å, which is within the predicted range (50–90 Å, see above) (Figure 4). Why have Neisseria adapted to attach respiratory proteins to the outer membrane via flexible tethers? To limit protein dynamics and enhance formation of electron-transfer complexes distance between adjacent amino acids along the peptide chain will be in the region between 1.5 and 3 Å (the translational distances between amino acids in the α-helix and in collagen respectively). From this, we estimate that the tethers can allow the globular domains of AniA, Laz and CCP to be ∼50–90 Å from the surface of the inner leaflet of the outer membrane. Hong et al. [18] showed that the Laz protein or a GST (glutathione transferase) fused to the signal peptide plus Nterminal LCR of Laz enables the globular azurin–GST to be surface-exposed in Neisseria (or indeed on expression in E. coli). Presumably, in this case, (at least some of) Laz is covalently linked to lipid in the outer leaflet of the outer membrane. The alternative explanation would be for the LCR to cross the outer membrane; this seems unfeasible, given that it is a highly charged peptide chain. Surface exposure of Laz (or other redox proteins) may be important for cell biological reasons, but it is hard to reconcile an external location for these outer membrane redox proteins with their function in respiration. There are no candidate electron-transporting outer membrane proteins in Neisseria that might enable electrons to pass from the periplasm out to an extracellularly located respiratory reductase, such as AniA or CCP. Thus we reason that a subpopulation of these outer membrane proteins may flip across to the outer leaflet of the outer membrane, but that the population that function in respiration are tethered to the inner leaflet of the outer membrane. A further reason for presuming that the AniA (and CCP) enzymes are located within the periplasm (although tethered to the outer membrane) lies in the overall similarity of their globular domains to their homologues from other organisms. There are no gross differences in the structure of AniA compared with other NirK homologues, indicating that it receives electrons via formation of a direct electrontransfer complex directly with a cytochrome or cupredoxin, as seen for its relatives from, e.g., Achromobacter xylosoxidans [19]. Any putative outer membrane pore that enabled this interaction would need to have an internal pore diameter of ∼30 Å to house a cytochrome/cupredoxin domain. In N. meningitidis, mutation of the gene encoding an inner membrane cytochrome c5 ablates nitrite reduction, supporting a role for this cytochrome as the sole electron In a system where redox proteins are free to move in the periplasm, there is effectively free tumbling in the aqueous milieu, although diffusion rates may be slow due to constrained space, since the compartment has a high concentration of biomolecules including the cross-linked cell wall polymer peptidoglycan. Tethering of a protein to the outer membrane will limit its capacity for lateral movement, as it would need to move both the membrane anchor and the globular protein domain in a way that would avoid becoming tangled in the peptidoglycan. Furthermore, and more importantly for electron transfer, tethering may reduce the capacity for tumbling. The AniA protein is a trimer [9], and thus in N. meningitidis, it is tethered to the outer membrane by three distinct peptides. The enzyme structure approximates to that of a triangular prism with a height of ∼50 Å and triangular sides of ∼70 Å. The three tethering peptides will thus prevent the enzyme from being able to rotate freely (because the tethers are not long enough to wrap around the enzyme). The tethers are attached to the face of the protein opposite the electron-accepting face, thus the tethers ensure that the AniA enzyme is maintained in a position in which the redox-accepting sites face the cytoplasmic membrane. The tethers may also thread through the peptidoglycan within the periplasm, further reducing mobility of the tethered enzyme. The cytochrome c5 electron donor is associated with the cytoplasmic membrane via a predicted membrane-spanning α-helix. The constrained nature of both donor and acceptor favour the formation of an electron-transfer complex. Essentially, the tethering lowers the loss of entropy that would be associated with the formation of a complex between a membrane-bound protein and a freely tumbling protein in aqueous solution, since, in the current example, both proteins already have a significantly deceased number of degrees of freedom compared with freely tumbling proteins. Thus there is a thermodynamic explanation for the tethering. The decreased dynamics of the proteins and the tendency to be appropriately oriented for complex formation should also translate into a kinetic advantage as the likelihood of an appropriate collision between the two partners is increased. The existence of tethered enzymes seems to be uncommon (but see below), and so there presumably is some C The C 2011 Biochemical Society Authors Journal compilation 1897 1898 Biochemical Society Transactions (2011) Volume 39, part 6 Figure 4 Arrangement of AniA and cytochrome c5 between the inner and outer membranes in Neisseria The AniA structure is based on the structure of AniA from N. gonorrhoeae [9]. The cytochrome c5 structure was modelled based on the structure of the di-haem cytochrome c4 from Pseudomonas stutzeri [27]. disadvantage to this organizational strategy. The limited dynamic movement of the AniA protein favours its interaction with cytochrome c5 as electron donor, but the disadvantage may be a lack of flexibility to form interactions with different electron-carrying partners in a flexible branched respiratory chain. Tethering of redox proteins for reasons related to Neisseria lifestyle An alternative to the biochemical arguments for redox protein tethering may relate to the lifestyle of Neisseria species and moonlighting functions for proteins such as Laz and AniA. Soluble periplasmic homologues of Laz, such as the azurin from Pseudomonas aeruginosa have been shown to have roles in apoptosis, in particular of cancer cells (see, e.g., [22]). N. meningitidis Laz has also been shown to be competent at entering glioblastoma cells and causing a high level of cytotoxicity [18]. This ability of Laz to enter mammalian cells is related to its lipid attachment and exposure to the surface of the cell [18]. Recently, it has been shown that antibodies that specifically recognize Laz (or another outer membrane protein Lip) are able to block serum-dependent killing of meningococcal cells, and that these antibodies recognize sequences in the LCR [23]. Evidence has also been presented that AniA (from N. gonorrhoeae) confers serum C The C 2011 Biochemical Society Authors Journal compilation resistance, via a mechanism that is independent of its nitrite reductase activity, indicating that this protein might also have a moonlighting role in subversion of the host immune response against Neisseria [24]. Tethered redox proteins outside Neisseria A standard organizational strategy in Gram-positive bacteria is the tethering of extracellular proteins such as homologues of redox proteins that are found in the periplasm in Gramnegative bacteria, and will not be discussed in the present paper. What is notable, however, is that bioinformatic searches reveal that tethering of redox proteins may also be fairly widespread among Gram-negative bacteria belonging to proteobacterial phylum. Nitrite reductases have been demonstrated to be commonly distributed by horizontal gene transfer [25,26]. Closely related to the Neisseria AniA nitrite reductases are the nitrite reductases from Gram-positive members of the Flavobacteria class, which is part of the Bacteroidetes phylum. These flavobacterial nitrite reductases are predicted to be membrane-associated lipoproteins, as would be expected for organisms with a single membrane. More interesting is that predicted lipid-tethered copper-type nitrite reductases are also found within the gammaproteobacterial genera Moraxella and Psychrobacter. Although there is no detectable ICoN2 and the NCycle16 conservation with the Neisseria LCR region, there is a region of 35 amino acids between the predicted N-terminal cysteine residue of the mature protein and the beginning of a recognizable nitrite reductase globular domain. The copper-type nitrite reductases from the gammaproteobacteria Moraxella and Psychrobacter are most closely related to those from the betaproteobacterium Neisseria, indicating that the outer membrane tethering may have arisen just once for this enzyme within the proteobacteria. Lipid-modified CCPs are also observed outside Neisseria species. CCP homologues within the deltaproteobacterial genera of Myxococcus, Stigmatella and Haliangium all contain a CCP with predicted N-terminal lipoprotein-attachment sites. Again the predicted N-terminal cysteine residue is followed by around 35 amino acids before the beginning of the CCP globular protein structure. The CCP enzymes from the Deltaproteobacteria are not particularly closely related to Neisseria CCP; closer relatives being found in such phyla as Aquificae and the cyanobacteria, suggesting that this strategy for localizing CCP may have arisen more than once. 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